Salmonella are mobile, gram-negative, rod-shaped bacteria. The taxonomic classification of Salmonella is based on their somatic (O) antigens and flagellar (H) antigens according to the Kaufmann-White system (a diagnostic antigen table) and results in a classification system in which Salmonella are declared as serovars and are characterized and classified on the basis of a sero formula. Of the more than 2400 Salmonella serovars known, only twenty to thirty are so far of importance in practice as pathogens in epidemiological diseases. These include the pathogens causing typhoid (S. typhi) and paratyphoid (S. paratyphi A, S. paratyphi B, S. paratyphi C) and a large number of enteritis pathogens, the so-called “enteritis Salmonella.” Whereas the typhoid and paratyphoid Salmonella cause serious generalized systematic infections, i.e., infections involving the entire body, an infection with enteritis Salmonella is usually limited locally to the intestines.
Salmonellosis in humans is usually a food-borne disease. The infection generally occurs due to consumption of infected or contaminated foods, whereas transmission to humans through direct contact with animals shedding Salmonella is rare. Direct or indirect human-to-human transfer may take place as a nosocomial infection in predisposed patients or under unfavorable hygiene conditions.
Primary sources of infection include in particular foods originating from poultry, cattle and swine, the animals themselves being sick only in extremely rare cases. Consequently, detection of the pathogens and/or antibodies plays a crucial role not only in human medicine but also in food operations and in veterinary medicine. The infectious dose for an adult human is 104 to 106 microorganisms. The incubation time is 5-72 hours and depends on the size of the infectious dose.
In a case of an enteritis Salmonella infection, a so-called enteritis salmonellosis, the infection usually manifests itself with diarrhea, nausea or vomiting and moderate fever. The symptoms usually last only a few hours or days. In weakened patients, however, this disease may also be fatal.
Shedding of enteritis Salmonella usually lasts an average of three to six weeks but in infants it may continue for several months.
In patients with a pre-existing congenital burden, Salmonella may trigger a reactive arthritis as a secondary illness 2-6 weeks after the enteritis infection; in rare cases, this arthritis may assume a chronic manifestation. At this point in time, no pathogens are usually detectable and/or culturable, so serological methods (Widal agglutination, see below) play a major role in detecting a past Salmonella infection (i.e., with manifestations in the past).
Under the German Infection Prevention Act, a tentative diagnosis or manifestation of acute infectious gastroenteritis must be reported under certain circumstances; ditto for detection of Salmonella. Furthermore, there are various legal requirements in Germany and other EU countries concerning required measures for combating Salmonella (e.g., Bovine Salmonella Regulations, Poultry Salmonella Regulations, various regulations of the feed and food law). In addition, various Salmonella monitoring programs have been established as part of self-monitoring in production.
The main point of emphasis in clinical laboratory diagnostic methods for salmonelloses is in culturing pathogens from fecal stool specimens and classifying them as suspected cases of Salmonella with the help of omnivalent and/or polyvalent Salmonella infectious sera. As a rule, such a tentative diagnosis can only be made approximately one to two days after receiving the sample in the diagnostic laboratory. In most cases another 2-3 days are needed for a confirmed diagnosis of Salmonella infection. In this period of time, suspected individual clones are characterized biochemically (colored series) and serologically. For serological differentiation, O and H antigens are analyzed in the form of microscope slide agglutination (Kaufmann-White system). This is done by first using polyvalent Salmonella test sera to determine the precise antigen formula with monovalent O and H antisera. As a rule, a total of 3-5 days are therefore needed for definitive detection of a Salmonella infection. In a case of salmonellosis caused by contaminated food in particular, the long amount of time required to obtain a diagnosis is a major problem because other people may become infected in the meantime. Therefore, in a suspected case of Salmonella infection, the source of infection must be localized and further dissemination prevented. In addition, patients are also isolated to prevent transmission of the infection. Early diagnosis is of great importance for the success of this measure.
Serological detection of Salmonella antibodies plays a major role in veterinary medicine and in the food industry, especially in the form of ELISA systems. For example, in Germany as well as in neighboring countries, the Salmonella antibody status (mainly anti-LPS immunoglobulin) of animal populations is currently being monitored in particular. Detection is performed on blood or meat juices. However, this method has limitations because it cannot reliably detect all pathogenic Salmonella serovars.
In human medicine however, especially in typhoid Salmonella infections, the so-called Widal agglutination test in particular is used to supplement bacteriological detection of the pathogen. In this test, patient serum is combined with boiled (O-antigen agglutination) or formalinized (H-antigen agglutination) Salmonella suspensions and tested for agglutination of the bacteria. One disadvantage of this is that not all infections are associated with the development of an Anti-O-Antigene titer; secondly, antibodies to H antigens may persist for many years after an infection. However, titer against O antigenes usually drop off again after a few weeks. It is thus impossible to reliably differentiate between an acute infection and an infection that has already been overcome.
Under in vitro culture conditions, Salmonella secretes a panel of proteins into the ambient medium. One of these proteins is the SipC protein.
The publications and other materials, including patents, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference in their entirety.
The nucleotide sequence of the SipC gene is known and is available to those skilled in the art via gene databanks (e.g., accession no.: U25631 or X82670). WO 03/000935 (see also U.S. Patent Publication 20030022214) discloses the use of PCR primers and FRET hybridization probes against the SipC gene for detection of Salmonella. However, this publication does not mention the use of SipC protein for the same purpose, in particular the use of antibodies to SipC protein.
In Weinrauch et al. “Neutrophil Elastase Targets Virulence Factors of Enterobacteria” (Nature 2002, 417, 6884, 91-4) and Hayward et al. “Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella” (EMBO J. 1999, 18, 18, 4926-34) the use of a polyclonal antibody to SipC protein for detection thereof in Immunoblot is described.
However, the polyclonal antibodies described there are not monospecific for Salmonella and therefore are not suitable for specific detection of Salmonella—as required especially in human diagnostics and in food testing.
In the context of the present invention, it has surprisingly been found (1.) that the SipC protein is a highly preserved molecule, i.e., there are only minor differences among the amino acid sequences of various Salmonella serovars, and (2.) that Salmonella already at a very early point in time produce the proteins of the typ-III-secretion system.
There is a need in the art for a Salmonella detection system that is capable of providing a rapid and reliable yes or no answer to the question of whether a certain sample is currently infected with Salmonella or whether there has been an infection in the past.